Busson, Sophie Louise; (2020) MarvelD3-mediated regulation of inflammatory stress signalling in epithelia. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Our understanding of tight junctions as signalling hubs is consistently gaining new ground. My thesis identifies and starts to define the regulation of signalling pathways central to cell and tissue physiology and pathology by the tight junction and transmembrane protein MarvelD3. In multiple epithelial cell lines, MarvelD3 is identified as a novel substrate of EGFR; direct EGFR-mediated phosphorylation of MarvelD3 Y404 occurs in response to EGF stimulation, which is confirmed through in vitro assays. Mutagenesis studies identifies this tyrosine as an integral component of a newly identified basolateral sorting signal and clathrin-coated pit consensus site, required for tight junction integration and clathrin-mediated endocytosis of MarvelD3, respectively. Regulation is bidirectional as MarvelD3 regulates EGFR activity, localisation, and expression, the latter by an Akt-dependent increase in gene methylation. In epithelia, breakdown of cell-cell adhesion and barrier function often elicits an inflammatory response, augmenting the detrimental effect on tissue integrity; however, it remains to be elucidated whether the tight junction plays a regulatory role in directing pro-inflammatory signalling. As such, loss- and gain-of-function approaches were employed, alongside small molecule inhibition, demonstrating that RNAi-mediated depletion of MarvelD3 induces activation of NFB and TCF signalling by a TBK1-dependent mechanism. This data was further supported by transcriptomic and proteomic analysis of pathway components. In addition, MarvelD3 regulates an Akt/mTOR signalling network via distinct modes: direct regulation of activity of key signalling components and expression of specific pathway components, which inhibitor and knockdown experiments reveal is dependent on PI3K and TBK1. Moreover, MarvelD3 depletion drastically downregulates 4E-BP1 expression in vitro and in vivo, in agreement with the induction of ER stress, the unfolded protein response, and giant vacuole formation in intestinal epithelial cells. My data implicate MarvelD3 as a sensor of tissue integrity that regulates signalling mechanisms important for cell and tissue maintenance and function.

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